The ultimate objective of our research is to understand the structure/function relationships of the components of the biochemical machinery for protein glycosylation and the molecular basis of their gene expression during animal growth and development. The focus of investigation is on the biosynthesis and regulation of N-linked glycoproteins in the mammary gland during its ontogeny. The biosynthesis of the precursor carbohydrate unit of these proteins is initiated by a stepwise, dolichol-linked assembly of the tri-branched Glc3Man9GlcNAC2-P-P-dolichol followed by its transfer en bloc to the nascent polypeptide in the RER. Subsequently, an extensive re-modeling of the oligosaccharide moiety occurs to give rise to completed glycoproteins. Glucosidases I and II are critically juxtapositioned in the post-translational maturation phase of N-linked glycoprotein synthesis since the sequential action of these two enzymes serves as a trigger for the oligosaccharide processing and polypeptide folding machinery for glycoprotein maturation in the secretory pathway of the cell. Inhibition of glucosidases I and II has been shown to interfere with normal folding, transport and egress of glycoproteins from the ER and cause accumulation and degradation of the malfolded glycoproteins. The impairment of oligosaccharide processing has been shown to affect the biological activity of many glycoproteins, transport of receptors of the cell surface, myoblast fusion, virus assembly and infectivity, reversal of the transformed phenotype of cells in vitro, and inhibition of tumor cell metastasis in vivo. Based on preliminary studies, we propose to pursue the following specific aims: 1 and 2. Identify the active site of amino acid residues and the catalytic nucleophile of glucosidases I and II by a combination of labeling with novel photoaffinity probes, conjugation with a suicide substrate and mutagenesis of selected evolutionary conserved nucleophiles and acidic residues in the enzymes; 3, Express catalytically active recombinant forms of Glucosidases I and II, and determine the crystal structure of the enzymes; 4. Investigate the significance of subunit interaction in the regulation of glucosidase II during the development and lactogenic differentiation of the mammary gland. N-linked glycoproteins, with diverse and versatile sugar moieties, represent the largest class of glycoproteins, participate in myriad biological phenomena, and are implicated in numerous pathologies, including malignancy, atherosclerosis, many genetic disorders and host-viral interaction leading to AIDS. Transgenic biotechnology can potentially provide the mammary gland as an excellent bioreactor for a 'molecular pharming' of glycoprotein pharmaceuticals.